Cysteine proteases play important roles in the biology of malaria parasites. We showed previously that cysteine protease activity is required for the hydrolysis of hemoglobin and for parasite development. Since our last renewal of this project, important advances have been made in our understanding of this class of enzymes, and in particular in the papain-family proteases known as falcipains. Most importantly, we now appreciate that there are multiple falcipains with important similarities and differences that suggest shared mechanisms, but also specific biological roles for each enzyme. Recent advances have included the heterologous expression of the hemoglobinases falcipain-2 and falcipain-3, characterization of the biochemical and biological features of these enzymes, characterization of falcipain homologs from other plasmodial species, identification of falcipain inhibitors of different chemical classes, and disruption of falcipain genes to begin to definitively determine enzyme functions. However, our understanding remains incomplete. At present, we cannot yet define specific roles for each falcipain, but powerful tools are available to critically assess their functions. Based on our studies of falcipains, we have generated hypotheses that will be tested in our ongoing project. We hypothesize that falcipains have multiple critical roles in the P. falciparum life cycle and that inhibition of these enzymes blocks parasite development by preventing these activities. More specifically, we hypothesize that falcipain-2, falcipain-2', and falcipain-3 have complementary, but not fully redundant roles in hemoglobin hydrolysis by erythrocytic parasites, that they also cleave erythrocytic cytoskeletal proteins to mediate the egress of merozoites, and that they are appropriate chemotherapeutic targets. We further hypothesize that distinct domains, some of which are unique to falcipains, direct enzyme trafficking, folding, processing, activity, and inhibition. The specific aims of our project will be: (1) evaluation of biochemical and biological properties of the falcipains, (2) characterization of the biological roles of falcipains with genetic studies, and (3) characterization of the biological roles of falcipains with specific inhibitors and biologically relevant substrates. Experiments designed to accomplish these aims should improve our understanding of the biology of malaria parasites and aid in the characterization of falcipains as potential drug targets.